EP0839830A1 - Acides nucléiques polyéthers - Google Patents
Acides nucléiques polyéthers Download PDFInfo
- Publication number
- EP0839830A1 EP0839830A1 EP97308707A EP97308707A EP0839830A1 EP 0839830 A1 EP0839830 A1 EP 0839830A1 EP 97308707 A EP97308707 A EP 97308707A EP 97308707 A EP97308707 A EP 97308707A EP 0839830 A1 EP0839830 A1 EP 0839830A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- group
- compound
- nucleobase
- dna
- linker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
Definitions
- the present invention relates in general to nucleotide mimics and their derived nucleic acid mimics, methods for the construction of both and the use of the nucleic acid mimics in biochemistry and medicine. More particularly, the present invention relates to (i) acyclic nucleotide mimics, also referred to as acyclic nucleotides; (ii) a method for synthesizing the acyclic nucleotide mimics; (iii) acyclic nucleotide mimic sequences, also referred to as acyclic polynucleotide sequences; (iv) a method for synthesizing the acyclic nucleotide mimic sequences; and (v) use of the acyclic nucleotide mimic sequences as oligonucleotides in for example antisesnse procedures.
- acyclic nucleotide mimics also referred to as acyclic nucleotides
- An antisense oligonucleotide may bind its target nucleic acid either by Watson-Crick base pairing or Hoogsteen and anti-Hoogsteen base pairing. To this effect see, Thuong and Helene (1993) Sequence specific recognition and modification of double helical DNA by oligonucleotides Angev. Chem. Int. Ed. Engl. 32:666.
- heterocyclic bases of the antisense oligonucleotide form hydrogen bonds with the heterocyclic bases of target single-stranded nucleic acids (RNA or single-stranded DNA), whereas according to the Hoogsteen base pairing, the heterocyclic bases of the target nucleic acid are double-stranded DNA, wherein a third strand is accommodated in the major groove of the B-form DNA duplex by Hoogsteen and anti-Hoogsteen base pairing to form a triplex structure.
- antisense oligonucleotides According to both the Watson-Crick and the Hoogsteen base pairing models, antisense oligonucleotides have the potential to regulate gene expression and to disrupt the essential functions of the nucleic acids. Therefore, antisense oligonucleotides have possible uses in modulating a wide range of diseases.
- oligonucleotides Since the development of effective methods for chemically synthesizing oligonucleotides, these molecules have been extensively used in biochemistry and biological research and have the potential use in medicine, since carefully devised oligonucleotides can be used to control gene expression by regulating levels of transcription, transcripts and/or translation.
- Oligodeoxyribonucleotides as long as 100 base pairs (bp) are routinely synthesized by solid phase methods using commercially available, fully automated synthesis machines. The chemical synthesis of oligoribonucleotides, however, is far less routine. Oligoribonucleotides are also much less stable than oligodeoxyribonucleotides, a fact which has contributed to the more prevalent use of oligodeoxyribonucleotides in medical and biological research, directed at, for example, gene therapy or the regulation of transcription or translation levels.
- Gene expression involves few distinct and well regulated steps.
- the first major step of gene expression involves transcription of a messenger RNA (mRNA) which is an RNA sequence complementary to the antisense (i.e., -) DNA strand, or, in other words, identical in sequence to the DNA sense (i.e., +) strand, composing the gene.
- mRNA messenger RNA
- mRNA messenger RNA
- transcription occurs in the cell nucleus.
- the second major step of gene expression involves translation of a protein (e.g., enzymes, structural proteins, secreted proteins, gene expression factors, etc.) in which the mRNA interacts with ribosomal RNA complexes (ribosomes) and amino acid activated transfer RNAs (tRNAs) to direct the synthesis of the protein coded for by the mRNA sequence.
- a protein e.g., enzymes, structural proteins, secreted proteins, gene expression factors, etc.
- ribosomal RNA complexes ribosomes
- tRNAs amino acid activated transfer RNAs
- RNA-synthesizing enzyme -- RNA polymerase an RNA-synthesizing enzyme -- RNA polymerase. This recognition is preceded by sequence-specific binding of one or more protein transcription factors to the promoter sequence. Additional proteins which bind at or close to the promoter sequence may upregulate transcription and are known as enhancers. Other proteins which bind to or close to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors.
- gene expression is typically upregulated by transcription factors and enhancers and downregulated by repressors.
- Most conventional drugs function by interaction with and modulation of one or more targeted endogenous or exogenous proteins, e.g., enzymes.
- Such drugs typically are not specific for targeted proteins but interact with other proteins as well.
- a relatively large dose of drug must be used to effectively modulate a targeted protein.
- Typical daily doses of drugs are from 10 -5 - 10 -1 millimoles per kilogram of body weight or 10 -3 - 10 millimoles for a 100 kilogram person. If this modulation instead could be effected by interaction with and inactivation of mRNA, a dramatic reduction in the necessary amount of drug could likely be achieved, along with a corresponding reduction in side effects. Further reductions could be effected if such interaction could be rendered site-specific. Given that a functioning gene continually produces mRNA, it would thus be even more advantageous if gene transcription could be arrested in its entirety.
- antisense or sense oligonucleotides or analogs that bind to the genomic DNA by strand displacement or the formation of a triple helix may prevent transcription.
- antisense oligonucleotides or analogs that bind target mRNA molecules lead to the enzymatic cleavage of the hybrid by intracellular RNase H.
- the oligonucleotides or oligonucleotide analogs provide a duplex hybrid recognized and destroyed by the RNase H enzyme.
- such hybrid formation may lead to interference with correct splicing.
- Chiang et al. (1991) Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms. J. Biol. Chem. 266:18162. As a result, in both cases, the number of the target mRNA intact transcripts ready for translation is reduced or eliminated.
- antisense oligonucleotides or analogs that bind target mRNA molecules prevent, by steric hindrance, binding of essential translation factors (ribosomes), to the target mRNA, as described by Paterson et al. (1977) Proc. Natl. Acad. Sci. USA, 74:4370, a phenomenon known in the art as hybridization arrest, disabling the translation of such mRNAs.
- antisense sequences which as described hereinabove may arrest the expression of any endogenous and/or exogenous gene depending on their specific sequence, attracted much attention by scientists and pharmacologists who were devoted at developing the antisense approach into a new pharmacological tool. To this effect see Cohen (1992) Oligonucleotide therapeutics. Trends in Biotechnology, 10:87.
- a c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G(0) to G(1). Nature, 328:445), reduced survival (Reed et al. (1990) Antisense mediated inhibition of BCL2 prooncogene expression and leukemic cell growth and survival: comparison of phosphodiester and phosphorothioate oligodeoxynucleotides. Cancer Res. 50:6565) and prevent receptor mediated responses (Burch and Mahan (1991) Oligodeoxynucleotides antisense to the interleukin I receptor m RNA block the effects of interleukin I in cultured murine and human fibroblasts and in mice. J. Clin. Invest. 88:1190).
- antisense oligonucleotides as antiviral agents the reader is referred to Agrawal (1992) Antisense oligonucleotides as antiviral agents. TIBTECH 10:152.
- the oligonucleotides or analogs must fulfill the following requirements (i) sufficient specificity in binding to the target sequence; (ii) solubility in water; (iii) stability against intra- and extracellular nucleases; (iv) capability of penetration through the cell membrane; and (v) when used to treat an organism, low toxicity.
- oligonucleotides are impractical for use as antisense sequences since they have short in vivo half-lives, during which they are degraded rapidly by nucleases. Furthermore, they are difficult to prepare in more than milligram quantities. In addition, such oligonucleotides are poor cell membrane penetraters, see, Uhlmann et al. (1990) Chem. Rev. 90:544.
- Oligonucleotides can be modified either in the base, the sugar or the phosphate moiety. These modifications include the use of methylphosphonates, monothiophosphates, dithiophosphates, phosphoramidates, phosphate esters, bridged phosphorothioates, bridged phosphoramidates, bridged methylenephosphonates, dephospho internucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether bridges, sulfoxy bridges, sulfono bridges, various "plastic" DNAs, ⁇ -anomeric bridges and borane derivatives. For further details the reader is referred to Cook (1991) Medicinal chemistry of antisense oligonucleotides - future opportunities. Anti-Cancer Drug Design 6:585.
- the recognition moieties are various natural nucleobases and nucleobase-analogs and the backbone moieties are either cyclic backbone moieties comprising furan or morpholine rings or acyclic backbone moieties of the following forms: where E is -CO- or -SO 2 -.
- the specification of the application provides general descriptions for the synthesis of subunits, for backbone coupling reactions, and for polymer assembly strategies.
- WO 86/05518 indicates that the claimed polymer compositions can bind target sequences and, as a result, have possible diagnostic and therapeutic applications, the application contains no data relating to the binding capabilities of a claimed polymer.
- the linking moiety in the oligonucleotide analogs is selected from the group consisting of sulfide (-S-), sulfoxide (-SO-), and sulfone (-SO 2 -).
- sulfide -S-
- SO- sulfoxide
- sulfone -SO 2 -
- PNAs peptide nucleic acids
- PNA oligomers can be synthesized from the four protected monomers containing thymine, cytosine, adenine and guanine by Merrifield solid-phase peptide synthesis. In order to increase solubility in water and to prevent aggregation, a lysine amide group is placed at the C-terminal.
- PNA PNA
- bind so strongly to target sequences they lack the specificity of their natural counterparts and end up binding not just to target sequences but also to other strands of DNA, RNA or even proteins, incapacitating the cell in unforeseen ways.
- oligonucleotide analogs devoid of these drawbacks which are characterized by (i) sufficient specificity in binding to target sequences; (ii) solubility in water; (iii) stability against intra- and extracellular nucleases; (iv) capability of penetrating through cell membranes; and (v) when used to treat an organism, low toxicity, properties that collectively render an oligonucleotide analog highly suitable as an antisense therapeutic drug.
- nucleotide mimics and their derived nucleic acid mimics having a polyether backbone methods for the construction of both and the use of the polyether nucleic acid mimics in biochemistry and medicine.
- a compound comprising a polyether backbone (i.e., the backbone itself consisting only C-C and C-O bonds) bearing a plurality of ligands that are individually bound to chiral carbon atoms located within the backbone, at least one of the ligands including a moiety selected from the group consisting of a naturally occurring nucleobase (i.e., native nucleobase, e.g., A, C, G, T, U), a nucleobase binding group (i.e., a moiety which is not a native nucleobase, yet as native nucleobases may form hydrogen bonds with nucleobases in a fashion similar to native nucleobases, e.g., inosine, thiouracil, bromothymine, azaguanines, azaadenines, 5-methylcytosine) and a DNA intercalator.
- a polyether backbone i.e., the backbone itself consisting only C-C and C-
- the chiral carbon atoms are separated from one another in the backbone by from four to six intervening atoms. Preferably five intervening atoms.
- the compound has the formula: or the formula: wherein, n is an integer greater than one, typically n is in the range of 5-20, preferably 7-15; each of B1 - Bn is a chemical functionality group, at least one of the B1 - Bn groups is a naturally occurring nucleobase, a nucleobase binding group or a DNA intercalator; each of Y1 - Yn is a first linker group; each of X1-Xn is a second linker group; C1 - Cn are chiral carbon atoms; and [K] and [I] are a first and second exoconjugates.
- each of the B1 - Bn chemical functionality groups is independently selected from the group consisting of a hydrogen group, a hydroxy group, an amino group, an amido group, a sulfhydril group, a carboxylic group, a (C1-C3) alkanoyl group, an aromatic group, a heterocyclic group, a chelating agent and a reporter group.
- each of the Y1 - Yn first linker groups is independently selected from the group consisting of an alkyl group, a phosphate group, a (C2-C4) alkylene chain, a (C2-C4) substitued alkylene chain and a single bond.
- each of the Y1 - Yn first linker groups is independently selected from the group consisting of a methylene group and a C-alkanoyl group.
- each of the X1 - Xn second linker groups is independently selected from the group consisting of a methylene group, an alkyl group, an amino group, an amido group, a sulfur atom, an oxygen atom, a selenium atom, a C-alkanoyl group, a phosphate derivative group, a carbonyl group and a single bond.
- m percents of the C1 - Cn chiral carbons are in an S configuration, wherein m is selected from the group consisting of 90-95 %, 96-98 %, 99 % and greater than 99 %.
- [K] and/or [I] are each a polyethylene glycol moiety.
- the compound has the formula:
- the compound is interacted with ions of an alkaline, earth alkaline or transition metal.
- a monomeric compound having a formula: wherein, B is a chemical functionality group; Y is a first linker group; X is a second linker group; C is chiral carbon atoms; Z is a first protecting group; and A is a leaving group.
- the B chemical functionality group of the monomeric compound is a naturally occurring nucleobase or a nucleobase binding group, should the nucleobase include an amino group, the amino group is protected by a second protecting group.
- the Z protecting group is selected from the group consisting of a dimethoxytrityl group, a trityl group, a monomethoxytrityl group and a silyl group.
- the A leaving group is selected from the group consisting of a halide group, a sulfonate group, an ammonium derivative and a radical moiety that could be replaced by SN1 or SN2 mechanisms (for SN1 or SN2 mechanisms see Roberts and Caserio (1965) Basic principles of organic chemistry. U. A. Benjamin Inc. New-York, NY, page 292).
- the second protecting group is selected from the group consisting of a benzamido group, an isobutyramido group, a t-butoxycarbonyl group, a fluorenylmethyloxycarbonyl group and an acid labile group which is not cleaved by reagents that cleave the Z protecting group.
- the monomeric compound has the formula:
- a process for preparing a compound according to the present invention comprising the step of sequentially condensing monomers each having an ether moiety, the ether moiety including at least one ether linkage, the ether moiety further including at least one chiral carbon atom to which a functionality group being linked, at least one of the functionality groups is selected from the group consisting of a naturally occurring nucleobase, a nucleobase binding group and a DNA intercalator.
- a process for preparing a compound according to the present invention comprising the step of sequentially condensing a first monomers having an ether moiety, the ether moiety including at least one ether linkage, the ether moiety further including at least one chiral carbon atom to which a functionality group being linked, with at least one additional monomer, wherein at least one of the functionality groups is selected from the group consisting of a naturally occurring nucleobase, a nucleobase binding group and a DNA intercalator.
- a process for sequence specific recognition of a double stranded polynucleotide comprising the step of contacting the polynucleotide with a compound according to the present invention, such that the compound binds in a sequence specific manner to one strand of the polynucleotide, thereby displacing the other strand.
- a process for sequence specific recognition of a single-stranded polynucleotide comprising the step of contacting the polynucleotide with a compound according to the present invention, such that the compound binds in a sequence specific manner to the polynucleotide.
- a process for modulating the expression of a gene in an organism comprising the step of administering to the organism a compound according to the present invention, such that the compound binds in a sequence specific manner DNA or RNA deriving from the gene.
- the modulation includes inhibiting transcription of the gene.
- the modulation includes inhibiting replication of the gene.
- the modulation includes inhibiting translation of the RNA of the gene.
- a process for treating conditions associated with undesired protein production in an organism comprising the step of contacting the organism with an effective amount of a compound according to the present invention, the compound specifically binds with DNA or RNA deriving from a gene controlling the protein production.
- a process for inducing degradation of DNA or RNA in cells of an organism comprising the steps of administering to the organism a compound according to the present invention, the compound specifically binds to the DNA or RNA.
- a process for killing cells or viruses comprising the step of contacting the cells or viruses with a compound according to the present invention, the compound specifically binds to a portion of the genome or to RNA derived therefrom of the cells or viruses.
- a pharmaceutical composition comprising a compound according to the present invention and at least one pharmaceutically effective carrier, binder, thickener, dilutent, buffer, preservative or surface active agent.
- the present invention successfully addresses the shortcomings of the presently known configurations by providing an oligonucleotide analog characterized by (i) sufficient specificity in binding its target sequence; (ii) solubility in water; (iii) stability against intra- and extracellular nucleases; (iv) capability of penetrating through the cell membrane; and (v) when used to treat an organism, low toxicity, properties collectively rendering the oligonucleotide analog of the present invention highly suitable as an antisense therapeutic drug.
- the present invention is of compounds that are not polynucleotides yet which bind to complementary DNA and RNA sequences
- the compounds according to the invention include naturally occurring nucleobases or other nucleobases binding moieties (also referred herein as nucleobase analogs) covalently bound to a polyether backbone, which can be used as oligonucleotide analogs in for example antisesnse procedures.
- the oligonucleotide analogs according to the present invention include a new acyclic biopolymer backbone which best fulfills the five criteria for selecting antisense oligonucleotide analogs listed in the background section above.
- the polyether poly(ethylene glycol) (PEG) is one of the best biocompatible polymers known, which possesses an array of useful properties. Among them, are a wide range of solubilities in both organic and aqueous media (Mutter et al. (1979) The Peptides Academic Press, 285), lack of toxicity and immunogenicity (Dreborg et al. (1990), Crit. Rev. Ther. Drug Carrier Syst. 6:315), nonbiodegradability, and ease of excretion from living organisms (Yamaoka et al. (1994) J. Pharm. Sci. 83:601).
- PEG conjugates For example, using PEG conjugates, immunogenicity and antigenicity of proteins can be decreased. To this effect see U.S. Pat. No. 4,179,337 to Davis et al. Thrombogenicity as well as cell and protein adherence can be reduced in the case of PEG-grafted surfaces. To this effect see Merrill (1992) Poly(ethylene Glycol) Chemistry, page 199, Plenum Press, Mew York. These beneficial properties conveyed by PEG are of enormous importance for any system requiring blood contact. For further information concerning the biocompatibility of PEG, the reader is referred to Zalipski (1995) Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates. Bioconjugate Chem. 6:150. However, all so far known PEG conjugates are exoconjugates, wherein the conjugated moiety is conjugated at one of the terminal hydroxyl groups of PEG (see formula I below).
- PEG is used, according to a preferred embodiment of the present invention, as a backbone to which nucleobases, nucleobase analogs (i.e., nucleobase binding moieties) and/or other chemical groups that interact with nucleic acids (e.g., DNA intercalators) are covalently linked to form oligonucleotide analogs having desired characteristics, as is further detailed below.
- the present invention provides a new class of acyclic backbone DNA compounds, that complementary bind single-stranded (ss) DNA and RNA strands.
- These compounds are herein referred to as polyether nucleic acids (ENAs).
- ENAs polyether nucleic acids
- the compounds of the invention generally include (i) a polyether backbone (i.e., a backbone consisting of only C-C and C-O bonds) and (ii) chemical functionality groups at least some of which are capable of forming suitable hydrogen bonds in a complementary manner with ssDNA and RNA.
- chemical functionality groups include either the five naturally occurring DNA and RNA nucleobases, i.e., thymine, adenine, cytosine, uracil or guanine, or modified bases such as but not limited to inosine, thiouracil, bromothymine, azaguanines, azaadenines, 5-methylcytosine, typically attached to a polyether backbone such as PEG via a suitable linker arm made of one or more linker groups, such that, in a preferred embodiment of the invention, adjacent chemical functionality groups are separated from one another by eleven atoms, mimicking native DNA.
- PEG is of a formula HO-(CH 2 CH 2 O) n -CH 2 CH 2 OH, repeated in (I):
- the polyether nucleic acid compound has the general formula (II): wherein, each of B1 - Bn is a chemical functionality group; each of Y1 - Yn is a first linker group; each of X1-Xn is a second linker group; C1 - Cn are chiral carbon atoms; and [K] and [I] are a first and second exoconjugates.
- the chemical functionality groups B1 - Bn are naturally occurring or analog nucleobases attached to the backbone in a predetermined selected order, forming a sequence.
- the nucleobases are attached to Y via the position found in nature, i.e., position 9 for purines (e.g., adenine and guanine), and position 1 for pyrimidines (e.g., uracyl, thymine and cytosine).
- some of the chemical functionality groups B1 - Bn may be a hydroxy group, an amino group, an amido group, a sulfhydril group, a carboxylic group, a (C1-C3) alkanoyl group, an aromatic group, a heterocyclic group, a chelating agent (e.g., EDTA, EGTA, a diol group such as a vicinal diol group, a triol group and the like).
- a chelating agent e.g., EDTA, EGTA, a diol group such as a vicinal diol group, a triol group and the like.
- some B1 - Bn functionality groups may be a DNA intercalator such as but not limited to an antraquinone group and the like.
- one or more of the functionality groups B1 - Bn may include a reporter molecule such as, for example, a fluorophor, a radioactive label, a chemiluminescent agent, an enzyme, a substrate, a receptor, a ligand, a hapten, an antibody and the like, such that the compound may serve as a labeled or detectable probe in hybridization assays.
- a reporter molecule such as, for example, a fluorophor, a radioactive label, a chemiluminescent agent, an enzyme, a substrate, a receptor, a ligand, a hapten, an antibody and the like, such that the compound may serve as a labeled or detectable probe in hybridization assays.
- any one or more of the B1 - Bn chemical functionality groups can be a ligand capable of interacting and covalently alter a complementary DNA or RNA strand.
- Suitable ligands include natural or analog nucleobase modified with an alkylating electrophile, such as but not limited to 3-(iodoacetamido)propyl, in position 5 of deoxyuridine.
- the modified compound may upon base pairing with a complementary target nucleic acid strand, to covalently cross link with the 7-position of a guanine residue present in the complementary DNA or RNA strands.
- Each of Y1 - Yn first linker groups can be an alkyl group such as a secondary carbon atom, a tertiary carbon atom or a phosphate group.
- each of the Y1 - Yn linker groups is a methylene group or a C-alkanoyl group.
- each of the Y1 - Yn linker groups can be a (C2-C4) alkylene chain or a (C2-C4) alkylene chain substituted with R 1 R 2 . In some cases Y can be just a single bond.
- Each of the X1-Xn second linker groups can be a methylene group (or carbon atom substituted with alkyl groups as R 1 R 2 ), an amino group, an amido group, a sulfur atom, an oxygen atom, a selenium atom, a C-alkanoyl group, a phosphate derivative group (e.g. methyl phosphate and phosphoamidate), or preferably a carbonyl group.
- X can be just a single bond
- the X and Y groups serve as linker arms to ensure the presence of preferably eleven atoms spacing between adjacent chemical functionality groups B, as is the case in natural nucleic acids.
- Figures la-b present two adjacent nucleobases (B) on a DNA strand ( Figure la) and on an ENA strand according to the preferred embodiment of the invention ( Figure 1b).
- C1 - Cn are chiral carbon atoms.
- the chirality of these atoms may be selected either of S or R configurations. Presently, the S configuration is preferred.
- the compound according to the invention is built in a stepwise manner, wherein each monomer or building block is sequentially added to a growing polymer. Therefore, provided that the building blocks can be prepared with a desired chirality (i.e., R or S configurations) a compound of predetermined yet mixed S and R configurations C1 - Cn chiral carbons can be prepared.
- [K] and [I] are a first and second exoconjugates such as but not limited to a polyethylene glycol (PEG) moieties each having one or more repeat units or a hydrogen atom.
- Exoconjugate [K] and [I] may be water soluble or water insoluble polymers. Such conjugates can be used to modulate the ability of the compound to cross cell membranes. Nevertheless, any one or both [K] and [I] may be a hydrogen atom.
- a preferred polyether nucleic acid molecule according to the invention have the general formula (III): wherein, each of B1 - Bn is a chemical functionality group such as a natural nucleobase or a nucleobase analog and PEG is polyethylene glycol.
- the most preferred embodiment is the compound having the above general formula III, wherein B is a natural nucleobase, i.e., thymine (T), adenine (A), cytosine (C), guanine (G) and uracil (U), and wherein n is an integer in the range of 4 to 50, preferably in the range of 8 to 30, most preferably in the range of 12-22.
- B is a natural nucleobase, i.e., thymine (T), adenine (A), cytosine (C), guanine (G) and uracil (U), and wherein n is an integer in the range of 4 to 50, preferably in the range of 8 to 30, most preferably in the range of 12-22.
- molecular modeling that represents the hybridization of a tetra-thymidine-ENA compound according to formula III above with natural adenine tetra nucleotide predicts a perfect hybridization match of the hydrogen bonds of the hybrid with minimum energy, wherein O is presented in red; C in yellow; N in blue, P in purple and the hydrogen bonds formed are emphasized by dashed lines, connecting the relevant atoms.
- polyether nucleic acids of the present invention may be synthesized using standard DNA synthesis procedures, either in solution or on a solid phase.
- the building blocks used are specially designed chiral (S) monomer triols or their activated forms.
- the monomer building blocks according to the invention are preferably triols or ketotriols having the general formula (IV): wherein B, Y and X are as defined above; Z is a suitable protecting group; and A is a suitable leaving group.
- a specific building block include B which is a natural or analog nucleobase
- the amino groups thereof may be protected with any conventional protecting group, such as but not limited to a benzamido group, an isobutyramido group, a t-butoxycarbonyl (Boc) group, a fluorenylmethyloxycarbonyl (Fmoc) group and the like.
- Z is a protecting group for protecting the terminal hydroxyl group of the monomer.
- Z can be any suitable protecting group known in the art, such as but not limited to a dimethoxytrityl group, a trityl group, a monomethoxytrityl group or a silyl group.
- Z is a dimethoxytrityl group.
- A is a leaving group such as a halide group, a sulfonate group, an ammonium derivative, or any radical moiety that could be replaced by SN1 or SN2 mechanisms.
- a preferred monomer building block according to the invention have the general formula (V): wherein, B, Z and A are as defined above.
- the polyether nucleic acid compound has the general formula (VI): wherein B1-Bn, Y1-Yn, X1-Xn, [K] and [I] are all as described above in detail.
- the difference between the compounds generally described by formula VI and the compounds generally described by formula II is in the number of atoms present between adjacent B functionality groups. While in the compound described by formula II present are eleven such atoms, in the compound described by formula VI present are only ten atoms, as the carbon at position 6 (or 5) as shown in Figure 1b is removed to yield an C-O-C bond.
- a polyether nucleic acid according to any of the embodiments described hereinabove is interacted with ions of an alkaline metal such as but not limited to Na + , earth alkaline metal such as but not limited to Ca ++ and Mg ++ , or ions of a transition metal such as but not limited to Fe ++ , Zn ++ , Cu ++ , Mn ++ and Cr ++ , capable of forming coordinative or other bonds with oxygen atoms or other electronegative moieties of the polyether backbone and/or the linker groups.
- an alkaline metal such as but not limited to Na +
- earth alkaline metal such as but not limited to Ca ++ and Mg ++
- ions of a transition metal such as but not limited to Fe ++ , Zn ++ , Cu ++ , Mn ++ and Cr ++
- Such coordinative bonds may assist in bringing the polyether nucleic acids according to the invention to a conformation highly suitable for base pairing with a complementary single-stranded DNA or RNA.
- Mg ++ ions are shown each to form three coordinative bonds with a polyether nucleic acid, two of which are with two adjacent oxygen atoms of the polyether backbone and one additional bond is formed with an oxygen atom of a carbonyl linker group.
- the present invention is further directed at use of ENA molecules in solid-phase biochemistry (see, Solid-Phase Biochemistry - Analytical and Synthetic Aspects (1983) W. H. Scouten, ed., John Wiley & Sons, New York), notably solid-phase biosystems, especially bioassays or solid-phase techniques which concerns diagnostic detection/quantitation or affinity purification of complementary nucleic acids (see, Affinity Chromatography - A Practical Approach (1986) P. D. G. Dean, W. S. Johnson and F. A. Middle, eds., IRL Press Ltd., Oxford; Nucleic Acid Hybridization - A Practical Approach (1987) B. D. Harnes and S. J. Higgins, IRL Press Ltd., Oxford).
- ENA species benefit from the above-described solid-phase techniques with respect to the much higher (and still sequence-specific) binding affinity for complementary nucleic acids and from the additional unique sequence-specific recognition of (and strong binding to) nucleic acids present in double-stranded structures. They can therefore replace common oligonucleotides in hybridization assays such as but not limited to blot hybridizations ("Southern” and “Northern”), dot blot hydridizations, reverse blot hybridizations, in situ hybridizations, liquid phase hybridizations, clones (bacteria/phages, etc.) screening and in other assays involving hybridizations such as but not limited to PCR, sequencing, primer extension and the like.
- hybridization assays such as but not limited to blot hybridizations ("Southern” and “Northern"), dot blot hydridizations, reverse blot hybridizations, in situ hybridizations, liquid phase hybridizations, clones (bacteria/
- the present invention is further directed at therapeutic and/or prophylactic uses for polyether nucleic acids (ENAs).
- ESAs polyether nucleic acids
- Likely therapeutic and prophylactic targets according to the invention include but are not limited to human papillomavirus (HPV), herpes simplex virus (HSV), candidia albicans, influenza virus, human immunodeficiency virus (HIV), intracellular adhesion molecules (ICAM), cytomegalovirus (CMV), phospholipase A2 (PLA2), 5-lipoxygenase (5-LO), protein kinase C (PKC), and RAS oncogene.
- HPV human papillomavirus
- HSV herpes simplex virus
- candidia albicans influenza virus
- HMV human immunodeficiency virus
- IAM intracellular adhesion molecules
- CMV cytomegalovirus
- PKA2 phospholipase A2
- Potential applications of such targeting include but are not limited to treatments for labial, ocular and cervical cancer; genital warts; Kaposi's sarcoma; common warts; skin and systemic fungal infections; AIDS; pneumonia; flu; mononucleosis; retinitis and pneumonitis in immunosuppressed patients; ocular, skin and systemic inflammation; cancer; cardiovascular disease; psoriasis; asthma; cardiac infarction; cardiovascular collapse; kidney disease; gastrointestinal disease; osteoarthritis; rheumatoid arthritis; septic shock; acute pancreatitis; and Crohn's disease.
- the polyether nucleic acids of the present invention can be formulated in a pharmaceutical composition, which may include thickeners, carriers, buffers, diluents, surface active agents, preservatives, and the like, all as well known in the art.
- Pharmaceutical compositions may also include one or more active ingredients such as but not limited to antiinflammatory agents, antimicrobial agents, anesthetics and the like in addition to polyether nucleic acids.
- the pharmaceutical composition may be administered in either one or more of ways depending on whether local or systemic treatment is of choice, and on the area to be treated. Administration may be done topically (including ophtalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, or intramuscular injection.
- Formulations for topical administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders.
- Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
- Coated condoms may also be useful.
- compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.
- Formulations for parenteral administration may include but are not limited to sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
- Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons ordinarily skilled in the art can easily determine optimum dosages, dosing methodologies and repetition rates.
- Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes transcription (including DNA-RNA transcription and reverse transcription), RNA transcripts or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the present invention. Seemingly diverse organisms such as yeast, bacteria, algae, protozoa, all plants and all higher animal forms, including warm-blooded animals, can be treated.
- each cell of multicellular eukaryotes can be treated since they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity.
- organelles e.g., mitochondria, chloroplasts and chromoplasts
- organelles e.g., mitochondria, chloroplasts and chromoplasts
- single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic phosphorothioate oligonucleotides.
- therapeutics is meant to include the eradication of a disease state, by killing an organism or by control of erratic or harmful cellular growth or expression.
- Polyether nucleic acids enjoy various advantages over existing oligonucleotide analog technologies.
- the ENAs' backbone is PEG, known to be soluble both in aqueous and in organic solvents, in high concentrations.
- the polyether backbone of ENAs according to the invention possess hydrophobicity on one hand and solubility in water on the other. This unique characteristic of ENAs enables a balanced hybridization between ENAs and complementary DNA or RNA molecules, as ENAs do not interact too strong with complementary sequences as protein nucleic acids (PNAs) do, yet ENAs are not highly solvated in aqueous media as native DNA and RNA strands.
- PNA-DNA hybrids are characterized by high melting temperature (Tm).
- Tm melting temperature
- the Tm value for a duplex such as PNA-T 10 -dA 10 is greater than 70 °C
- Tm value of the equivalent native double stranded DNA (dT 10 -dA 10 ) is nearly three fold lower, about 24 °C.
- body temperature e.g., 37 °C
- PNAs lack the specificity to their intended counterparts and end up binding not just to target sequences but also to other strands of DNA, RNA, or even proteins, incapacitating the cell in unforeseen ways.
- PNAs act as a micelle when the lysine residues are solvated. PNAs are poorly miscible in water, while the hydrophobic nature of the backbone have a tendency to seek for a nonpolar environment e.g., the bases of the natural complementary DNA. These hydrophobic interactions are the major driving force for the formation of highly stable PNA-DNA hybrid and therefore very high Tm values for such hybrids.
- the unique solubility nature of ENAs by conserving the hydrophobic-hydrophilic properties of polyethers such as PEG, yield Tm values slightly higher than natural DNA, yet much lower values than PNAs, which moderate values are of great importance for specificity.
- polyether compounds such as cyclodextrins have a tendency to form helices, which are stabilized in solution by water and metal ions under physiological conditions. This phenomenon is schematically illustrated for an ENA compound according to the invention in Figure 4. This characteristic of polyether compounds renders these compounds highly suitable acyclic backbones for nucleobases to be base paired with complementary DNA or RNA molecules.
- PEG is approved by the FDA for parenteral use, topical application, and as a constituent of suppositories, nasal sprays, foods and cosmetics.
- PEG is of low toxicity when administered orally or parenterally, and only large quantities involve adverse reactions. See, Smyth, H. F. et al. (1955) J. Am. Pharm. Assoc., 34:27.
- ENAs include a PEG backbone and/or are conjugated to PEG exoconjugates and therefore enjoy the above listed advantages.
- ENAs synthesis preferably involves using monomers having one chiral center with known chirality.
- This monomer (formula V) is condensed as much as needed to prepare the appropriate oligonucleotide having a polyether backbone and a preselected and desirable nucleobases sequence. During these condensations, the chiral center is not susceptible to racemization.
- the synthesis of the monomers involves a chiral starting material which is available in a desired chirality in a pure form. In contrast, Miller et al. (1971) J. Am. Chem. Soc.
- each methylphosphonate linkage (p) may have an R or an S chiral configuration.
- dApA(S)(dA) 12 hybridized to poly dT has a Tm value higher by 4.4 °C as compared with dApA(R)(dA) 12 .
- This observation suggests that the methyl groups in the R configuration may provide some specific steric hindrance.
- d(CpT) 8 is soluble up to millimolar concentrations, whereas d(ApG) 8 has solubility of less than 0.1 mM.
- the starting material for synthesizing a monomer according to formula V above is preferably (S)-(+)-Erythrulose hydrate (Aldrich). This compound has a chiral center which possesses the appropriate S configuration. Protection of the vicinal diols as acetonide is performed by dissolving 13.8 grams of (S)-(+)-Erythrulose hydrate in 11 ml acetic acid. The solvent is then removed by co-evaporation with 22 ml of added toluene.
- the obtained residue is dissolved in 10 % of 1,2-dimethoxypropane in 30 ml acetone containing a catalytic amount (1.9 grams) of p-toluenesulfonic acid. The mixture is stirred at room temperature for 30 minutes. 1.6 grams of sodium acetate are then added, the mixture is filtered and the obtained residue is chromatographed on a silica gel using ethylacetate/hexane (3/7) as the chromatographic carrier to afford Ca. 8.0 grams (50%) of 3,4- O -isopropylidene-(+)-erythrulose ( compound A ) as an oil.
- the carbonyl group of the 3,4- O -isopropylidene-(+)-erythrulose may be protected as dithian by reacting 3,4- O -isopropylidene-(+)-erythrulose with excess of methyl-trimethylsilylthiol (The later reagent is prepared according to Evans et al. (1977) J. Am. Chem. Soc. 99:5009), and Znl 2 in ether. The reaction is monitored on a TLC plate. At the end of the reaction, concentrated ammonia is added, and the resulting product is extracted with ether.
- the dithian compound thus obtained (compound B) is preferably purified by silica gel column chromatography using ethylacetate/hexane (3/7) as the eluent.
- Attachment of a nucleobase to (S)-4- O -methanesulfonyl-1,2-3-dithian butanetriol ( compound C ) :
- the following description refers to attachment of adenine, yet as will be appreciated any other native or analog nucleobase may be similarly attached.
- a mixture of 8.1 l grams (60 mmoles) of adenine and 2.4 grams of a 60 % NaH dispersion (60 mmoles) in 200 ml of DMF is stirred for 90 minutes at room temperature.
- 50 mmoles of compound C are added and the mixture is stirred for further 2.5 hours at 90 °C.
- a mixture of 11 mmoles of compound F is co-evaporated with dry pyridine and is thereafter dissolved in 100 ml of same.
- the mixture obtained is cooled in ice-water bath, and 13.2 mmoles of dimethoxytrityl chloride (Aldrich) dissolved in 75 ml of dry pyridine is added dropwise.
- the mixture is kept at room temperature for 17 hours, afterwhich the mixture is evaporated to dryness and extracted with ethylacetate/water (1:1), washed once with 100 ml saturated NaHCO 3 solution, twice with water and twice with brine solution.
- the organic layer is dried over anhydrous sodium sulfate, evaporated and purified by column chromatography using CH 2 Cl 2 /MeOH (95/5) to yield compound G.
- the preferred polymeric support for solid phase ENA synthesis is a Merrifield's peptide resin, 2% cross-linked (chloromethylated styrene/divinylbenzene copolymer, Aldrich).
- the chloride groups covalently attached to the polymer via CH 2 - groups are replaced by hydroxyl groups, by mixing 10 grams of the polymer with large excess (50 ml) of acetic acid/triethylamine (1/1) and absolute ethanol (100 ml), and heating the mixture at reflux temperature for 48 hours.
- the polymer is washed with ethanol under vacuum, and resuspended along with 5 grams of KOH in 100 ml methanol for 3 hours, at room temperature.
- the resulted hydroxy polymer is washed first with water then by methanol and is dried by washing with ether.
- the hydroxy polymer is allowed to react with 1,4-dibromo-2,3-isopropylidene butanediol by the following reactions.
- 1,4-dibromo-2,3-isopropylidene-2,3-butanediol is prepared by 1,4-dibromo-2,3-isopropylidene-2,3-butanediol .
- 1,4-dibromo-2,3-butanediol (24.8 grams, 100 mmoles, Aldrich) is dissolved in 200 ml dry acetone, containing a catalytic amount (1.9 grams, 10 mmoles) of p-toluenesulfonic acid. The mixture is stirred at room temperature for 3 hours. After addition of 1.6 grams sodium acetate (20 mmoles), the mixture is filtered and the residue is chromatographed on silica gel using CH 2 Cl 2 /hexane (9/1) to afford 27 grams (93%) as an oil.
- the resulting hydroxy group of the polymer thus obtained is ready to be condensed with the bromide of compound H, which is the preferred building block V of the present invention.
- the cycle of ENA synthesis includes three steps: condensation, capping and deprotection.
- Acetylation of unreacted polymer hydroxy groups is achieved by adding 10 ml of acetic anhydrid/lutidine/tetrahydrofuran (1/1/8) to the polymeric support resulted from the previous step. The suspension is agitated for five minutes. Then, the solvent is sucked by vacuum, washed twice with 10 ml methanol and twice with 10 ml dichloromethane.
- DMT dimethoxytrityl group
- the dried polymer is then condensed with a second compound following formula V to which a second base (B 2 ) is attached (e.g., compound H- B 2 ) in dry DMF in a manner as described above under condensation.
- a second base e.g., compound H- B 2
- Such cycles are repeated as much as needed to form appropriate antisense sequence, wherein in each tri-stages cycle one additional monomer is sequentially added to the growing chain.
- the polymeric support to which the antisense sequence is attached is treated with concentrated ammonium hydroxide for 16 hours at 55°C.
- the polymeric support is washed with water, methanol and with ether.
- the polymeric support resulted from the previous step is treated with a solution of 80% aqueous acetic acid and agitated at room temperature for 21 hours and for 4 hours at 50°C.
- the polymer is washed with water, methanol and ether.
- the polymeric support is further treated with a solution of one gram sodium periodate in 10 ml water for 3 hours.
- one gram of solid sodiumborohydride is added and the solution is stirred for one hour at room temperature.
- the solution is collected and purified by dialysis against water for 16 hours at 4 °C, followed by HPLC purification.
- PEG-NH 2 amino-polyethyleneglycol
- PEG Poly(ethylene glycol)
- ADA adenine deaminase
- Covalent attachment of PEG to ENA requires activation of the hydroxyl terminal group of the PEG polymer with a suitable leaving group that can be displaced by nucleophilic attack with nucleophiles.
- Activation of PEG could be achieved for example by converting its terminal hydroxyl group into a leaving amino group to obtain PEG-NH 2 .
- PEG can be converted into PEG-NH 2 by the following way (equations 15-17): (15) PEG-O-CH 2 -CH 2 -OH + Tosyl-chloride ⁇ PEG-O-CH 2 -CH 2 -O-Tosyl (16) PEG-O-CH 2 -CH 2 -O-Tosyl + NaN 3 ⁇ PEG-O-CH 2 -CH 2 -N 3 (17) PEG-CH 2 -CH 2 -N 3 + H 2 /Pd(C) ⁇ PEG-O-CH 2 -CH 2 -NH 2
- the PEG-NH 2 derivative can be condensed with carboxylic activated groups to form an amid linkage connecting an ENA molecule according to the invention with PEG, according to equation 18:
- the PEG-NH 2 derivative can alternatively be condensed with an aldehyde to form a Shiff base, which condensation is followed by reduction to an amino linkage connecting an ENA molecule according to the invention with PEG, according to equation 19:
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Polyethers (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/740,516 US5908845A (en) | 1996-10-30 | 1996-10-30 | Polyether nucleic acids |
US740516 | 1996-10-30 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0839830A1 true EP0839830A1 (fr) | 1998-05-06 |
EP0839830B1 EP0839830B1 (fr) | 2003-01-22 |
Family
ID=24976832
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97308707A Expired - Lifetime EP0839830B1 (fr) | 1996-10-30 | 1997-10-30 | Acides nucléiques polyéthers |
Country Status (5)
Country | Link |
---|---|
US (1) | US5908845A (fr) |
EP (1) | EP0839830B1 (fr) |
JP (1) | JP4108801B2 (fr) |
CA (1) | CA2217780C (fr) |
DE (1) | DE69718600T2 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6326479B1 (en) | 1998-01-27 | 2001-12-04 | Boston Probes, Inc. | Synthetic polymers and methods, kits or compositions for modulating the solubility of same |
Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7135191B2 (en) * | 1997-09-04 | 2006-11-14 | Zsolt Istvan Hertelendy | Urogenital or anorectal transmucosal vaccine delivery system |
CA2323929C (fr) | 1998-04-03 | 2004-03-09 | University Of Iowa Research Foundation | Procedes et produits servant a stimuler le systeme immunitaire au moyen d'oligonucleotides et de cytokines immunotherapeutiques |
US6348583B1 (en) * | 1999-08-30 | 2002-02-19 | Bio-Rad Laboratories, Inc. | Poly(ether-thioether), poly(ether-sulfoxide) and poly(ether-sulfone) nucleic acids |
ATE465739T1 (de) * | 2001-01-29 | 2010-05-15 | Bio Rad Laboratories | Nukleinsäurederivative |
US7052840B2 (en) * | 2002-04-03 | 2006-05-30 | Capitol Genomix, Inc. | Reversible association of nucleic acid with a carboxylated substrate |
US20090264635A1 (en) | 2005-03-25 | 2009-10-22 | Applera Corporation | Methods and compositions for depleting abundant rna transcripts |
BRPI0721095B1 (pt) | 2006-12-13 | 2015-09-29 | Luminex Corp | Sistemas e métodos para a análise multíplex de pcr em tempo real |
US9605035B2 (en) | 2008-01-10 | 2017-03-28 | Research Development Foundation | Vaccines and diagnostics for the ehrlichioses |
CN102264898B (zh) | 2008-10-23 | 2013-10-16 | 国立大学法人东京大学 | 微小rna的功能抑制方法 |
WO2010054328A2 (fr) | 2008-11-07 | 2010-05-14 | Research Development Foundation | Compositions et méthodes pour l'inhibition de la formation et de la signalisation du complexe cripto/grp78 |
US20110045080A1 (en) * | 2009-03-24 | 2011-02-24 | William Marsh Rice University | Single-Walled Carbon Nanotube/Bioactive Substance Complexes and Methods Related Thereto |
EP2789689B1 (fr) | 2009-06-29 | 2016-04-27 | Luminex Corporation | Amorces chimères avec des conformations en épingle à cheveux et leurs procédés d'utilisation |
US10028902B2 (en) | 2013-11-08 | 2018-07-24 | Baylor Research Institute | Nuclear localization of GLP-1 stimulates myocardial regeneration and reverses heart failure |
WO2016134293A1 (fr) | 2015-02-20 | 2016-08-25 | Baylor College Of Medicine | Inactivation de p63 pour le traitement de l'insuffisance cardiaque |
BR112018007611A2 (pt) | 2015-10-14 | 2018-10-23 | Bio Path Holding Inc | ácidos nucleicos p-etóxi para formulação lipossômica |
US20190177391A1 (en) | 2016-03-31 | 2019-06-13 | Baylor Research Institute | Angiopoietin-like protein 8 (angptl8) |
ES2928654T3 (es) | 2016-09-16 | 2022-11-21 | Bio Path Holdings Inc | Terapia combinada con oligonucleótidos antisentido liposomales |
US11479769B2 (en) | 2017-03-17 | 2022-10-25 | National University Corporation Chiba University | Technique for treating cancer using structurally-reinforced S-TuD |
EP4242316A4 (fr) | 2020-11-04 | 2024-09-04 | Synplogen Co Ltd | Plasmide intégratif |
CA3200833A1 (fr) | 2020-11-04 | 2022-05-12 | Synplogen Co., Ltd. | Production de plasmide vecteur viral dans bacillus subtilis |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1986005518A1 (fr) * | 1985-03-15 | 1986-09-25 | James Summerton | Polymeres stereoreguliers de liaison a un polynucleotide |
US4766062A (en) * | 1984-05-07 | 1988-08-23 | Allied Corporation | Displacement polynucleotide assay method and polynucleotide complex reagent therefor |
WO1996011205A1 (fr) * | 1994-10-06 | 1996-04-18 | Isis Pharmaceuticals, Inc. | Conjugues d'acides nucleiques peptidiques |
WO1996015778A1 (fr) * | 1994-11-18 | 1996-05-30 | Supratek Pharma, Inc. | Composes polynucleotidiques |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5216141A (en) * | 1988-06-06 | 1993-06-01 | Benner Steven A | Oligonucleotide analogs containing sulfur linkages |
US5608046A (en) * | 1990-07-27 | 1997-03-04 | Isis Pharmaceuticals, Inc. | Conjugated 4'-desmethyl nucleoside analog compounds |
US5688941A (en) * | 1990-07-27 | 1997-11-18 | Isis Pharmaceuticals, Inc. | Methods of making conjugated 4' desmethyl nucleoside analog compounds |
DK51092D0 (da) * | 1991-05-24 | 1992-04-15 | Ole Buchardt | Oligonucleotid-analoge betegnet pna, monomere synthoner og fremgangsmaade til fremstilling deraf samt anvendelser deraf |
US5519134A (en) * | 1994-01-11 | 1996-05-21 | Isis Pharmaceuticals, Inc. | Pyrrolidine-containing monomers and oligomers |
US5637684A (en) * | 1994-02-23 | 1997-06-10 | Isis Pharmaceuticals, Inc. | Phosphoramidate and phosphorothioamidate oligomeric compounds |
-
1996
- 1996-10-30 US US08/740,516 patent/US5908845A/en not_active Expired - Lifetime
-
1997
- 1997-10-29 CA CA002217780A patent/CA2217780C/fr not_active Expired - Lifetime
- 1997-10-30 JP JP31286697A patent/JP4108801B2/ja not_active Expired - Lifetime
- 1997-10-30 DE DE69718600T patent/DE69718600T2/de not_active Expired - Fee Related
- 1997-10-30 EP EP97308707A patent/EP0839830B1/fr not_active Expired - Lifetime
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4766062A (en) * | 1984-05-07 | 1988-08-23 | Allied Corporation | Displacement polynucleotide assay method and polynucleotide complex reagent therefor |
WO1986005518A1 (fr) * | 1985-03-15 | 1986-09-25 | James Summerton | Polymeres stereoreguliers de liaison a un polynucleotide |
WO1986005519A1 (fr) * | 1985-03-15 | 1986-09-25 | James Summerton | Reactif et procede d'analyse de polynucleotides |
WO1996011205A1 (fr) * | 1994-10-06 | 1996-04-18 | Isis Pharmaceuticals, Inc. | Conjugues d'acides nucleiques peptidiques |
WO1996015778A1 (fr) * | 1994-11-18 | 1996-05-30 | Supratek Pharma, Inc. | Composes polynucleotidiques |
Non-Patent Citations (1)
Title |
---|
CLOAD S T ET AL: "POLYETHER TETHERED OLIGONUCLEOTIDE PROBES", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 113, no. 16, 31 July 1991 (1991-07-31), pages 6324 - 6326, XP000570387 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6326479B1 (en) | 1998-01-27 | 2001-12-04 | Boston Probes, Inc. | Synthetic polymers and methods, kits or compositions for modulating the solubility of same |
Also Published As
Publication number | Publication date |
---|---|
DE69718600D1 (de) | 2003-02-27 |
CA2217780A1 (fr) | 1998-04-30 |
DE69718600T2 (de) | 2003-11-20 |
CA2217780C (fr) | 2007-10-23 |
JP4108801B2 (ja) | 2008-06-25 |
JPH10257888A (ja) | 1998-09-29 |
US5908845A (en) | 1999-06-01 |
EP0839830B1 (fr) | 2003-01-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0839830B1 (fr) | Acides nucléiques polyéthers | |
CA2382631C (fr) | Acides nucleiques de poly(ether-thioether), de poly(ether-sulfoxide) et de poly(ether-sulfone) | |
US6191266B1 (en) | Sugar modified nucleosides | |
US7683164B2 (en) | Nucleic acid derivatives | |
EP1015469B2 (fr) | Analogues d'oligonucleotides | |
EP0736093B1 (fr) | ACIDES NUCLEIQUES ANTI-SENS DESTINES A LA PREVENTION ET AU TRAITEMENT DE TROUBLES DANS LESQUELS INTERVIENT L'EXPRESSION DE c-erbB-2 | |
AU2002230058A1 (en) | Nucleic Acid Derivatives | |
US5703223A (en) | Solid phase synthesis of oligonucleotides with stereospecific substituted phosphonate linkages by pentavalent grignard coupling | |
US6743902B1 (en) | Sugar modified nucleosides | |
WO1993015742A1 (fr) | Oligonucleotides possedant des fractions zwitterioniques | |
WO1993023570A1 (fr) | Oligonucleotides ayant des conjugues fixes a la position 2' de la fraction sucre | |
AU2002325599B2 (en) | Oligonucleotide analogues | |
AU3144593A (en) | Oligonucleotides having aminohydrocarbon phosphonate moieties |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): DE FR GB |
|
AX | Request for extension of the european patent |
Free format text: AL;LT;LV;RO;SI |
|
17P | Request for examination filed |
Effective date: 19981102 |
|
AKX | Designation fees paid |
Free format text: DE FR GB |
|
RBV | Designated contracting states (corrected) |
Designated state(s): DE FR GB |
|
17Q | First examination report despatched |
Effective date: 20001213 |
|
GRAG | Despatch of communication of intention to grant |
Free format text: ORIGINAL CODE: EPIDOS AGRA |
|
GRAG | Despatch of communication of intention to grant |
Free format text: ORIGINAL CODE: EPIDOS AGRA |
|
GRAG | Despatch of communication of intention to grant |
Free format text: ORIGINAL CODE: EPIDOS AGRA |
|
GRAH | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOS IGRA |
|
GRAH | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOS IGRA |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): DE FR GB |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REF | Corresponds to: |
Ref document number: 69718600 Country of ref document: DE Date of ref document: 20030227 Kind code of ref document: P |
|
ET | Fr: translation filed | ||
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
26N | No opposition filed |
Effective date: 20031023 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: DE Payment date: 20081022 Year of fee payment: 12 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20081014 Year of fee payment: 12 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20081021 Year of fee payment: 12 |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: ST Effective date: 20100630 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FR Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20091102 Ref country code: DE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20100501 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20091030 |